Process For The Fermentative Preparation Of Sulphur-Containing Amino Acids

ABSTRACT

The invention relates to a process for the fermentative preparation of sulphur-containing amino acids chosen from the group of L-methionine, L-cysteine, L-cystine, L-homocysteine and L-homocystine, comprising the steps:
         a) provision of a microorganism of the family Enterobacteriaceae or of a microorganism of the family Corynebacteriaceae which has an increased thiosulphate sulphurtransferase activity compared with the particular starting strain;   b) fermentation of the microorganism, from a) in a medium which contains an inorganic source of sulphur chosen from the group of salt of dithiosulphuric acid or a mixture of a salt, of dithiosulphuric acid and a salt of sulphuric acid, a fermentation broth being obtained, and   c) concentration of the sulphur-containing amino acid in the fermentation broth from b).

The invention relates to a process for the fermentative preparation ofsulphur-containing amino acids chosen from the group of L-methionine,L-cysteine, L-cystine, L-homocysteine and L-homocystine.

Sulphur-containing amino acids are of great economic importance.L-Cysteine is used as a food additive, as a starting substance forpharmacological active compounds (e.g. N-acetylcysteine) and forcosmetics. The amino acid L-methionine plays a prominent role in animalnutrition. It belongs to the essential amino acids which cannot beproduced by biosynthesis in the metabolism of vertebrates. In animalbreeding it must consequently be ensured that adequate amounts ofmethionine are taken in with the feed. However, since L-methionine isoften present in conventional feedstuff plaints (such as soya orcereals) in amounts which are too low to ensure optimum animalnutrition, especially for pigs and poultry, it is advantageous to admixmethionine as an additive to the animal feed. D-Methionine can beconverted into biologically active L-methionine by vertebrates. Aracemate of D- and L-methionine is therefore usually added to the animalfeed, L-Homocysteine can be converted into L-methionine by animals bytransmethylation and can therefore replace this.

In the prior art, amino acids such as methionine are prepared bychemical synthesis. In this preparation, acrolein and methylmercaptanare first reacted to give 3-methylthiopropionaldehyde, which in turnwith cyanide, ammonia and carbon monoxide leads to hydantoin. This canfinally be hydrolysed to the racemate, an equimolar mixture of the twostereoisomers D- and L-methionine. Since the biologically active form ofthe molecule represents exclusively the L form, the D form contained inthe feed must first be converted, into the active L form in themetabolism by de- and transamination.

In contrast to methionine, most other natural, proteinogenic amino acidsare chiefly prepared by fermentation by microorganisms. This utilizesthe fact that microorganisms have appropriate biosynthesis pathways forsynthesis of the natural amino acids. In addition, many fermentationprocesses achieve very favourable production costs with inexpensiveeducts, such as glucose and mineral salts, and moreover deliver thebiologically active L form of the particular amino acid.

However, biosynthesis pathways of amino acids are subject to strictmetabolic control in wild-type strains, which ensures that the aminoacids are produced only for the cell's own. requirement. An importantprerequisite for efficient production processes is therefore thatsuitable microorganisms are available which, in contrast to thewild-type organisms, have a drastically increased production output, forthe preparation of the desired amino acid.

Such microorganisms which overproduce amino acids can be produced byconventional mutation/selection processes and/or by modern, targeted,recombinant techniques (metabolic engineering). In the latter case,genes or alleles which effect an amino acid overproduction by theirmodification, activation or inactivation are first-identified. Thesegenes/alleles are then introduced into a microorganism strain orinactivated by molecular biology techniques, so that an optimumoverproduction is achieved. However, often only the combination ofseveral different measures leads to a truly efficient production.

In E. coli and C. glutamicum, L-cysteine is derived biochemically fromL-serine. L-Serine is activated as O-acetylserine by the serineacetyltransferase by f. The O-acetylserine (thiol)lyase then transfersreduced sulphur in the form of sulphide, as a result of which L-cysteineis formed. In contrast to C. glutamicum, E. coli has two differentO-acetylserine (thiol) 1 vases , the O-acetylserine (thiol)lyase B(“CysM”) also being able to transfer thiosulphate to O-acetylserine. Asa result, S-sulphocysteine is formed, which is then split intoL-cysteine and sulphite or sulphate in a manner which has not yet beencharacterized (Kredich N. M. (1996) in Neidhardt FC et al. (ed.)“Escherichia coil and Salmonella”, 2nd edition, p. 514-527).

L-Methionine, together with lysine and threonine, is derived fromaspartate. Sulphur is introduced in the form of L-cysteine (viacystathionine as an intermediate product) into L-methionine bytranssulphurization (in C. glutamicum and E. coli; only route in E.coli). In parallel with this, e.g. in C. glutamicum there is the routeof direct sulfhydrylation, in which sulphide is assimilated in the formof L-homocysteine (B-J Hwang, H-J Yeom, Y Kim, H-S Lee, 2002, JBacteriol., 134 (5):1277-1286). The C1 group of the L-methionineoriginates from the C1 metabolism and it is transferred toL-homocysteine by the methionine synthases MetE and MetH (Review: GreeneR C (1996) in Neidhardt FC et al. (ed.) “Escherichia coli andSalmonella”, 2nd edition, p. 542-560). L-Methionine biosynthesis in C.glutamicum is described by Riickert et al. (Ruckert C, Punier A,Kalinowski J, 2003, J Biotechnol., 104(1-3):213-28). Strains andprocesses for the fermentative production of L-methionine have beendescribed e.g. for E. coli (WO2006/001616, WO2009/043803) and C.glutamicum (WO2009/144270).

The use of thiosulphate as a source of sulphur allows significantlyhigher theoretical yields in the production of sulphur-containing aminoacids (compared with sulphate) (Krömer J O, Wittmann. C, Schroder H,Heinzle E, Metab Eng., 2006, 8(4), pp. 353-369; and WO2007/020295). Theadvantage of thiosulphate is explained as follows: In sulphate (SO₄ ²⁻)the sulphur atom is present in oxidation level +6. For assimilation, itmust be reduced to oxidation level −2 (sulphide=S²⁻). For reduction of asulphate to sulphide, the cell must use 2 ATP and 4 NAD PH. (=8electrons) . In thiosulphate, the central sulphur atom has the oxidationlevel +5, and the terminal, sulphur atom has the oxidation level −1(average formal oxidation level of the two sulphur atoms: +2). Forreduction of both the sulphur atoms of thiosulphate, only 8 electronsare therefore required, compared with 16 electrons in the reduction of 2sulphates.

The use of thiosulphate as a source of S for the production ofL-cysteine with E. coli is described e.g. in DE 10 2007 007 333 andWO2001/27307.

It has been shown in WO2007/077041 that L-methionine production in E.coli can also be improved significantly by the use of thiosulphateinstead of sulphate.

It has not yet hitherto been shown experimentally that the use ofthiosulphate also leads to an. improvement in L-methionine production inC. glutamicum. It is shown in WO2007/020295 (p. 240) that with Nathiosulphate as a source of sulphur, C, glutamicum forms more biomass.This was evaluated as an indication of the lower ATP and NADPHconsumption. 1-Methionine production with thiosulphate was notinvestigated in practice.

In E. coli, thiosulphate is taken up by the sulphate/thiosulphatetransporter CysPUWA-Sbp (Kredich N. M. (1996) in Neidhardt F C et al.(ed.) “Escherichia coli and Salmonella”, 2nd edition, p. 514-527). CysPand Sbp are two different periplastic binding proteins. CysP has ahigher affinity for thiosulphate and Sbp has a higher affinity forsulphate. CysA forms the ATP-binding component, CysU and CysW are thetransmembrane components. In WO2009/043803, the expression of thecysPUWAM operon was amplified in E. coli and an improvement in.L-methionine production was thereby achieved. Nothing is known of theuptake of thiosulphate in C. glutamicum. Knock out mutants of theputative sulphate uptake system CysZ (cg3112) can still grow withthiosulphate as the source of S (Rückert C. et al. (2005; BMC Genomics.,vol. 6(121)). CysZ is therefore not (solely) responsible for transportof thiosulphate.

O-Acetylserine (thiol)lyase B (CysM, EC 2.5.1.47) makes it possible forE. coli to use thiosulphate as a source of S for the synthesis ofL-cysteine and L-methionine. The enzyme catalyses the formation ofS-sulphocysteine (R—S—SO₃) and acetate from O-acetylserine andthiosulphate. S-sulphocysteine is then cleaved into L-cysteine andsulphite or sulphate in a manner which has not hitherto beencharacterized (Kredich N. M. (1996) in Neidhardt FC et al. (ed.)“Escherichia coli and Salmonella”, 2nd edition, p. 514-527). Thesulphate is reduced to sulphite (SO₃ ²⁻) in. three steps by ATPsulphurylase (CysDN), APS kinase (CysC) and PAPS sulphotransferase(CysH). Sulphite is then reduced further by NADPH sulphite reductase togive sulphide (S²⁻), which can be used by O-acetylserine (thiol) lyase A(CysK) and G-acetylserine (thiol)lyase B (CysM) for synthesis of asecond L-cysteine molecule.

C. glutamicum can grow in minimal medium with thiosulphate as a sourceof sulphur (Rückert C. et al. (2005), BMC Genomics., vol. 6(121)) .Nevertheless, C. glutamicum has no O-acetylserine (thiol) lyase B(“CysM”) (Rückert and Kalinowski (2006) in A. Burkovski (ed.)“Corynebacteria: Genomics and. Molecular Biology”, Caister AcademicPress). Thiosulphate must therefore be assimilated in another manner.

The object of the present invention is to provide a process andmicroorganism strains which render possible a higher overproduction ofsulphur-containing amino acids, in particular of L-methionine.

This object is achieved by a process for the fermentative preparation ofsulphur-containing amino acids chosen from the group of L-methionine,L-cysteine, L-cystine, L-homocysteine and L-homocystine, comprising thesteps:

-   -   a) provision of a microorganism of the family Enterobacteriaceae        or of a microorganism of the family Corynebacteriaceae which has        an increased thiosulphate sulphurtransferase activity compared        with the particular starting strain;    -   b) fermentation, of the microorganism from a) in a medium which        contains a salt of dithiosulphuric acid or a mixture of a salt        of dithiosulphuric acid and a salt of sulphuric acid as an        inorganic source of sulphur, a fermentation broth being        obtained, and    -   c) concentration of the sulphur-containing amino acid in the        fermentation broth from b).

The invention furthermore provides a process for the fermentativepreparation of sulphur-containing amino acids chosen from the group ofL-methionine, L-cysteine, L-cystine, L-homocysteine and L-homocystine,comprising the steps:

-   -   a) provision of a microorganism of the family Enterobacteriaceae        or of a microorganism of the family Corynebacteriaceae which        overexpresses a gene coding for a polypeptide with the activity        of a thiosulphate sulphurtransferase;    -   b) fermentation of the microorganism from, a) in a medium which        contains a salt of dithiosulphuric acid or mixture of a salt of        dithiosulphuric acid and a salt of sulphuric acid as an        inorganic source of sulphur, a fermentation broth being        obtained, and    -   c) concentration of the sulphur-containing amino acid in the        fermentation broth from b).

Thiosulphate sulphurtransferases, also called “rhodaneses” (EC 2.8.1.1),are enzymes which transfer the reduced sulphur atom from thiosulphate tocyanide (CN″) .

Some thiosulphate sulphurtransferases can also transfer the reducedsulphur atom to alternative substrates, e.g. dihydrolipoate (AlexanderK., Volini M, 1987, J. Biol. Chem., 262: 6595-6604). In the database“Clusters of Orthologous Groups of proteins” (COG), 168 homologoussulphurtransferases in all three domains of life are currently to befound, in category COG0607 “Rhodanese-related sulphurtransferase”(Tatusov R L, Fedorova N D, Jackson J D, Jacobs A R, Kiryutin B, KooninE V, Krylov D M, Mazumder R, Mekhedov S L, Nikolskaya A N, Rao B S,Smirnov S, Sverdlov A V, Vasudevan S, Wolf Y I, Yin J J, Natale D A,2003, BMC Bioinformatics, 4:41; http://www.ncbi.nlm.nih.gov/COG/).Rhodaneses have one or more characteristic rhodanese-like domains (PFAMdatabase PF00581, http://pfam.sanger.ac.uk/; Gliubich F, Gazerro M,Zanotti G, Delbono S, Bombieri G, Berni R, 1996, J Biol Chem.,271(35):21054-61).

The gene RDL2 (rhodanese-like protein) from Saccharomyces carevisiaeS288c codes for the protein Rd12p 149 amino acids long. It has arhodanese-like domain (PFAM database PF00581,http://pfam.sanger.ac.uk/). It has been shown by experiment that Rd12pis a thiosulphate sulphurtransferase (rhodanese, EC 2.8.1.1; Foster M W,Forrester M T, Stamier J S, 2009, Proc Natl Acad Sci USA,106(45):18948-53) . SEQ ID NO: 1 shows the DNA sequence of the geneRDL2. SEQ ID NO: 2shows the amino acid sequence of the protein Rd12pcoded by RDL2.

E. coli has, in addition to the well-characterized rhodaneses GlpE andPspE (Adams K, Teertstra W, Koster M, Tommassen J, 2002, FEBS Lett. 518:173-6), seven further paralogous enzymes with rhodanese-like domains(Cheng H, Donahue J L, Battle S E, Ray W K, Larson T J, 2008, OpenMicrobiol J., 2:18-28):

YgaP has an N-terminal rhodanese-like domain and two transmembranedomains. It shows in vitro rhodanese activity (Ahmed, F., 2003,Dissertation).

YbbB is a tRNA 2-selenouridine synthase and has an N-terminalrhodanese-like domain (Wolfe M D, Ahmed F, Lacourciere G M, Lauhon C T,Stadtman T C, Larson T J, 2004, J Biol Chem., 279(3):1801-1809).

ThiI is necessary for synthesis of thiamine and plays a role in theconversion of uridine into thiouridine at position 8 in tRNA (PalencharP M, Buck C J, Cheng H, Larson T J, Mueller E G, 2000, J Biol Chem.,275(12):8283-8286). SseA is a 3-mercaptopyruvate:cyanidesulphurtransferase, which preferentially transfers 3-mercaptopyruvateinstead of thiosulphate to cyanide (Colnaghi R, Cassinelli G, DrummondM, Forlani F, Pagani S, 2001, FEBS Lett., 500(3):153-156). The enzymehas two rhodanese-like domains.

The ORF ynjE codes for a putative sulphurtransferase with threerhodanese-like domains (Hänzelmann P, Dahi J U, Kuper J, Urban A,Müller-Theissen U, Leimkühler S, Schindelin H. , 2009, Protein Sci.,18(12):2480-2491) .

The ORF yibN codes for a putative sulphurtransferase with a C-terminalrhodanese-like domain.

The ORF yceA codes for a putative sulphurtransferase with arhodanese-like domain.

In Corynebacterium glutamicum, at least 7 ORFs code for presumedsulphurtransferases:

The ORF thtR (=cg0803, NCgl0671) codes for a putative sulphurtransferasewith two rhodanese-like domains and a length of 301 amino acids.

The ORF sseA2 (=cg1613, NCgl1369) codes for a putativesulphurtransferase with two rhodanese-like domains and a length of 289amino acids.

The ORF cg3000 (=NCgl2616) codes for a putative sulphurtransferase witha rhodanese-like domain and a length of 96 amino acids.

The ORF cg0073 (=NCgl0053) codes for a putative sulphurtransferase witha rhodanese-like domain and a length of 97 amino acids.

The ORF cg0074 (=NCgl0054) codes for a purative sulphurtransferase witha rhodanese-like domain and a length of 197 amino acids.

The ORF sseA1 (=cg3073, NCgl2678) codes for a putativesulphurtransferase with two rhodanese-like domains and a length of 274amino acids.

The ORF cg3319 (=NCgl2890) codes for a putative sulphurtransferase witha rhodanese-like domain and a length of 312 amino acids.

The thiosulphate sulphurtransferase from cattle (Bos taurus) has tworhodanese-like domains and is well-characterized (Cannella C, Costa M,Pensa B, Ricci G, Pecci L, Cavallini D., 1981, Eur J Biochem.,119(3):491-495).

In a preferred embodiment of the process, the microorganismoverexpresses one or more gene(s) coding for a polypeptide with theactivity of a thiosulphate sulphurtransferase, the polypeptide with theactivity of a thiosulphate sulphurtransferase being chosen from thefollowing a) to d):

-   -   a) a polypeptide consisting of or containing the polypeptides        Rd12p, GlpE, PspE, YgaP, ThiI, YbbB, SseA, YnjE, YceA, YibN,        NCgl0671, NCgl1369, KCgl2616, NCgl0053, KCgl0054, NCgl2678,        NCgl2890; thiosulphate sulphurtransferase from, mammals, for        example the thiosulphate sulphur transferase from the bovine        liver (Bos taurus); preferably Rdl2p, GlpE, PspE and        particularly preferably Rdl2p;    -   b) a polypeptide consisting of or containing the amino acid        sequence shown in SEQ ID NO: 2;    -   c) a polypeptide with an amino acid sequence which is identical        to the extent of 70% or more to the amino acid sequence of a) or        b), the polypeptide having thiosulphate sulphur transferase        activity;    -   d) a polypeptide which has an amino acid sequence containing a        deletion, substitution, insertion and/or addition of from 1 to        45 amino acid residues with respect to the amino acid sequence        shown in SEQ ID NO: 2, the polypeptide having thiosulphate        sulphurtransferase activity.

As mentioned above, polypeptides with the activity of a thiosulphatesulphurtransferase also include variants of the enzymes mentioned undera) or b), with an amino acid sequence which is identical to the extentof 70% or more to the amino acid sequence of the sequences mentionedunder a) or b), the variants having thiosulphate sulphurtransferaseactivity. Preferred embodiments include variants which are at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98% orat least 99% identical to the amino acid sequences described, above,i.e. wherein at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% of the amino acid positions areidentical to those of the amino acid, sequences described above. Thepercentage identity is preferably calculated over the total length ofthe amino acid or nucleic acid region, A number of programs based on alarge number of algorithms are available to the person skilled in theart for sequence comparison. In this connection, the algorithms ofNeedleman and Wunsch or Smith and Waterman give particularly reliableresults. For the alignment of the sequences, the program PileUp (J. Mol.Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153)or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48;443-453 (1970)) and Smith and Waterman (Adv. Appl. Math, 2; 482-489(1981))], which belong to the GCG software package [Genetics ComputerGroup, 575 Science Drive, Madison, Wis., USA 53711 (1991)] areavailable. The percentage values given above for the sequence identityare preferably calculated over the total sequence region with the GAPprogram.

Polypeptides with the activity of a thiosulphate sulphurtransferasefurthermore also include fragments of the enzymes mentioned, in a) orb). These fragments have the activity described above. A fragment of apolypeptide with the activity of a thiosulphate sulphurtransferase inthe context of this patent application preferably contains at least 30,at least 50, or at least 80 successive amino acid residues of one of theabovementioned amino acid sequences.

As mentioned above, polypeptides with the activity of a thiosulphatesulphurtransferase also include variants of the enzymes mentioned undera) or b) which have an amino acid sequence containing a deletion,substitution, insertion and/or addition of from 1 to 45 amino acidresidues with respect to the amino acid sequence shown, in SEQ ID NO: 2,the polypeptide having thiosulphate sulphurtransferase activity. Inpreferred embodiments, the amino acid sequence contains a deletion,substitution, insertion and/or addition of from 1 to 40, furtherpreferably from 1 to 30, still further preferably from 1 to 20,preferably from 1 to 15, still further preferably from 1 to 10 and most,preferably from 1 to 5 amino acid, residues with respect to the aminoacid sequence shown in SEQ ID NO: 2.

In a further preferred process, the polypeptide is coded by a gene whichincludes the nucleotide sequence of SEQ ID NO: 1, which corresponds tothe wild-type sequence of the gene RDL2 from Saccharomyces cerevisiaeS288c

It is furthermore preferable for the polypeptide to be coded by a genewhich includes the nucleotide sequence of SEQ ID NO: 3. The codon usagehere is slightly adapted to that of E. coli.

In a further preferred process, the polypeptide is coded by a gene whichincludes the nucleotide sequence of SEQ ID NO: 4. The codon usage hereis more highly adapted to that of E. coli.

It is furthermore preferable for the polypeptide to be coded, by a genewhich includes the nucleotide sequence of SEQ ID NO: 5, The codon usagehere is adapted completely to chat of E. coli.

In carrying out the process according to the invention, the expressionof the gene coding for a polypeptide with the activity of a thiosulphatesulphurtransferase is preferably increased by one or more of thefollowing measures:

-   -   a) The expression of the gene is under the control of a promoter        which, in the microorganism used for the process, leads to an        amplified expression compared with, the starring strain. In this        context, for example, a constitutive GAPDK promoter of the gapA        gene of Escherichia coli, or an inducible lac, tac, trc, lambda,        ara or Let promoter can be used (Review; MaKrides S C.        Micro-biol Rev. 1996 September; 60(3):512-38) .    -   b) The number of copies of the gene coding for a polypeptide        with the activity of a thiosulphate sulphurtransferase is        increased compared with the starting strain. This can be        achieved, for example, by inserting the gene into plasmids with        an increased number of copies, or by integrating the gene into        the chromosome of the microorganism in several copies (Baneyx F,        1999, Curr. Opin. Biotechnol. 10, 411-421).    -   c) The expression of the gene is effected using a ribosome        binding site, which leads to an increased translation in the        microorganism used for the process compared with the starting        strain (Makrides S C. Microbiol Rev. 1996        September;60(3):512-38) .    -   d) The expression of the gene is amplified by optimization of        the codon usage of the gene with respect to the microorganism        used for the process (Welch, Vilalobos, Gustafsson and Minshull,        J R Soc Interface, 2009, 6: 3467-76), The codon adaptation index        (CAI), for example, is suitable as a measure of the adaptation        of the codon usage of a gene to an organism. (Sharp P M, Li W H,        1987, Nucleic Acids Res., 15(3):1281-95).    -   e) The expression of the gene is amplified by reduction of mRNA        secondary structures in the mRMA transcribed by the gene (Kuala        G, Murray A W, Tollervey D, Plotkin J B., Science, 2009 Apr. 10;        324(5924):255-8; Welch, Villalobos, Gustafsson and Minshull, J R        Soc Interface, 2009, 6:S467-76).    -   f) The expression of the gene is amplified by elimination of RNA        polymerase terminators in the mRNA transcribed by the gene        (Welch, Villalobos, Gustafsson and Minshull, J R Soc Interface,        2009, 6:S467-76).    -   g) The expression of the gene is effected using mRNA-stabilizing        sequences in the mRNA transcribed by the gene (Carrier T A,        Keasling J D, Biotechnol Prog., 1997, November-December; 13        (6):699-708) . The sequence ARNmst17 may be mentioned as an        example for stabilizing the mRNA of the gene metF of E. coli        (WO2009/043803),

In a further embodiment of the process according to the invention, it ispreferable for the salt of dithiosulphuric acid to be a salt chosen fromthe group of alkali metal salt, alkaline earth metal salt, ammonium saltand mixtures thereof, preferably ammonium salt.

It is furthermore preferable for the salt of sulphuric acid to be a saltchosen from the group of alkali metal salt, alkaline earth metal salt,ammonium salt (and mixtures), preferably ammonium salt.

Preferably, the concentration of the salt of dithiosulphuric acid in themedium or in the fermentation broth is 0.05 g/kg to 100 g/kg, preferably0.1 g/kg to 20 g/kg and particularly preferably 0.2 g/kg to 12 g/kg.

In a preferred process, the concentration of the salt of dithiosulphuricacid in the medium or in the fermentation broth during the fermentationis kept at at least 0.05 g/kg to 100 g/kg, preferably 0.1 g/kg to 20g/kg and particularly preferably 0.2 g/kg to 12 g/kg.

In a further preferred process, Curing the fermentation the content ofthe salt of dithiosulphuric acid, based on the total content ofinorganic sulphur in the medium and in the fermentation broth, is keptat at least 5 mol %.

The process according to the invention can be designed as a batchprocess, fed batch process, repeated fed batch process and continuousprocess.

The sulphur-containing amino acid can furthermore be obtained from thefermentation broth as a solid produce or in dissolved form in a liquidproduct.

In a preferred process of the present invention, the sulphur-containingamino acid to be produced is L-methionine.

It is furthermore preferable for the microorganism to be the genusEscherichia, particularly preferably the species Escherichia coli.

In an alternative process, the microorganism is the genusCorynebacterium, particularly preferably the species Corynebacteriumglutamicum.

In a particularly preferred process of the present invention, themicroorganism is chosen from:

-   -   Corynebacterium glutamicum with increased activity and/or        expression of aspartate kinase and attenuation or deletion of        the regulator protein McbR compared with the starting strain;

Escherichia coli with increased activity and/or expression of aspartatekinase and attenuation or deletion of the regulator protein MetJcompared with the starting strain.

The invention furthermore provides a microorganism chosen from

-   -   Corynebacterium glutamicum with increased activity and/or        expression of aspartate kinase and attenuation or deletion of        the regulator protein McbR compared with the starting strain;    -   Escherichia coli with increased activity and/or expression of        aspartate kinase and attenuation or deletion of the regulator        protein Mete compared with the starting strain;        wherein the microorganism secretes or produces L-methionine, and        wherein the microorganism has an increased thiosulphate        sulphurtransferase activity compared with the particular        starting strain.

The invention moreover provides a microorganism, chosen from

-   -   Corynebacterium glutamicum with increased activity and/or        expression of aspartate kinase and attenuation or deletion of        the regulator protein McbR compared with the starting strain;    -   Escherichia coli with increased activity and/or expression of        aspartate kinase and attenuation or deletion of the regulator        protein MetJ compared with the starting strain;        wherein the microorganism secretes or produces L-methionine, and        wherein the microorganism, overexpresses a gene coding for a        polypeptide with the activity of a thiosulphate        sulphurtransferase,

The strain of C. glutamicum which secretes or produces L-methioninepreferably has an increased enzyme activity of aspartate kinase (EC2.7.2.4), feedback-resistant alleles being preferred. In theCorynebacterium, this aspartate kinase is coded by the gene lysC. Due tothe attenuation or deletion of the regulator protein McbR (which iscoded by the gene mcbR), an increase in the sulphur utilisationfurthermore takes place, McbR is the repressor of the entire sulphurutilization cascade in C. glutamicum (Rey D A, Nentwich S S, Koch D J,Rückert C, Pühler A, Tauch A, Kalinowski J., Mol Microbiol., 2005, 56(4): 871-887).

The strain of E. coli which secretes or produces L-methionine preferablyhas an increased enzyme activity of aspartate kinase (EC 2.7.2.4),feedback-resistant alleles being preferred. In E. coli there are threedifferent aspartate kinases which are coded by the genes thrA, metL orlysC. Due to the attenuation or deletion of the regulator protein MetJ,which is coded by the gene metJ, an increase in the L-methioninebiosynthesis furthermore takes place. Met J is the main repressor ofL-methionine biosynthesis in E. coli.

It is furthermore preferable for this microorganism to overexpress oneor more gene(s) coding for a polypeptide with the activity of athiosulphate sulphurtransferase, the polypeptide with the activity of athiosulphate sulphurtransferase being chosen from the following a) tod):

-   -   a) a polypeptide consisting of or containing the polypeptides        Rdl2p, GlpE, PspE, YgaP, ThiI, YbbB, SseA, YnjE, YceA, YibN,        NCgl0671, NCgl1369, NCgl2616, NCgl0053, NCgl0054, NCgl2678,        NCgl2890; thiosulphate sulphurtransferase from mammals, for        example the thiosulphate sulphurtransferase from the bovine        liver (Bos taurus); preferably Rdl2p, GlpE, PspE and        Particularly preferably Rdl2p;    -   b) a polypeptide consisting of or containing the amino acid        sequence shown in SEQ ID NO: 2;    -   c) a polypeptide with an amino acid sequence which is identical        to the extent of 70% or more to the amino acid sequence of a) or        b), the polypeptide having thiosulphate sulphurtransferase        activity;    -   d) a polypeptide which has an amino acid sequence containing a        deletion, substitution, insertion and/or addition of from 1 to        45 amino acid residues with respect to the amino acid, sequence        shown in SEQ ID NO: 2, the polypeptide having thiosulphate        sulphurtransferase activity.

The starting strain of the microorganism furthermore is preferablyderived from the group consisting of Escherichia coli MG1655,Escherichia coli W3110, Escherichia coli DH5α, Escherichia coli DH10B,Escherichia coli BW2952, Escherichia coli REL606, Corynebacteriumglutamicum ATCC13032, Corynebacterium glutamicum R, Corynebacteriumglutamicum DSM20411 (former name Brevibacterium flavum), Corynebacteriumglutamicum DSM20412 (former name Brevibacterium lactofermentum),Corynebacterium glutamicum DSM1412 (former name Brevibacteriumlactofermentum), Corynebacterium efficiens YS-314^(T) (=DSM44549),Corynebacterium glutamicum ATCC21608, Corynebacterium glutamicumDSM17322.

A further strain which secretes or produces L-methionine is, forexample, the E. coli production strain MG1655 ΔmetJ Ptrc-metH Ptrc-metFPtreF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP containing the productionplasmid pME101-thrA*1-cysE-Pgap-metA*11 (WO2009/043803).

Cloning of the E. coli production strain MG1655ΔmetJ Ptrc-metH Ptrc-metFPtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP is described in the patentapplication WO2009/043803. The strain is based on the wild-type strainE. coli K12 MG1655. The following modifications have been introduced inthe genome of this strain:

-   -   The gene for the repressor to L-methionine biosynthesis metJ has        been deleted.    -   Upstream of the gene metH (codes for cobalamin-dependent        methionine synthase), the potent trc promoter has been inserted.    -   Upstream of the gene metF (codes for        5,10-methylenetetrahydrofolate reductase), the potent trc        promoter has been inserted,    -   Upstream of the operon cysPUWAM, the potent trcF promoter has        been inserted. cysPUWA codes for a sulphate/thiosulphate uptake        transporter. cysM codes for cysteine synthase B.    -   Upstream of the operon cysJIH, the potent trcF promoter has been        inserted. cysJI codes for sulphite reductase and cysH codes for        3′-phosphoadenylyl sulphate reductase.    -   Upstream of the operon gcvTHP, the potent trc09 promoter has        been inserted. gcvT, gcvH and gcvP code for three components of        the glycine cleavage system.

Cloning of the E. coli production plasmidpME101-thrA*1-cysE-Pgap-metA*11 is described in the patent applicationWO2007/077041. It is a plasmid with a low number of copies (low copyplasmid) based on the vector pCL1920 (Lerner, C. G. and Inouye, M.,Nucl, Acids Res. (1990)18: 4631 [PMXD: 2201955]). The empty plasmidpME101 has the lacI^(q) gene, which codes for a highly expressed alleleof the lac repressor. The gene thra*1 was cloned downstream of a potenttrc promoter which can be repressed by the Lac repressor. It codes for afeedback-resistant variant of aspartate kinase/homoserine dehydrogenaseThrA from E. coli. In the same orientation after it lies the gene cysEtogether with its natural promoter. It codes for serineacetyltransferase from E. coli. Downstream of cysE, the potent gapApromoter from E. coli follows, which controls the expression of the genemetA*11. metA*11 codes for a feedback-resistant variant of homoserineO-succinyltransferase from E. coli.

The following strains may be mentioned as examples of furthermicroorganisms which secrete or produce L-methionine:

-   -   C. glutamicum M11179 (=DSM17322) (WO2007/011939)    -   E. coli TF4076BJF metA#10+metYX(Lm) (WO2008/127240; page 46);    -   E. coli W3110AJ/pKP4 51 (EP 1 445 310 B1, page 7 ex. 4)    -   E. coli WΔthrBCΔmetJmetK32 pMWPthrmetA4Δ5Δ9 (Yoshihiro Usuda and        Osamu Kurahashi, 2005, Applied and Environmental Microbiology,        vol. 71, no, 6, p. 3228-3234)    -   W3110/pHC34 (WO01/27307 page 13, ex. 3).

Further examples of various suitable microorganisms are described byGomes et al, (Enzyme and Microbial Technology 37 (2005), 3-18).

In further preferred embodiments, the bacteria which produceL-methionine have one or more features chosen from the group of:

-   -   1) overexpressed polynucleotide which codes for one or more        components of the throsulphate/sulphate transport system CysPUWA        (EC 3.6.3.25),    -   2) overexpressed polynucleotide which codes for a        3′-phosphoadenosine 5′-phosphosulphate reductase CysH (EC        1.8.4.8),    -   3) overexpressed polynucleotide which codes for one or snore        components of the sulphite reductase CysJI (EC 1.8.1.2),    -   4) overexpressed polynucleotide which codes for a cysteine        synthase A CysK (EC 2.5.1.47),    -   5) overexpressed polynucleotide which codes for a cysteine        synthase B CysM (EC 2.5.1.47),    -   6) overexpressed polynucleotide which codes for a serine        acetyltransferase CysE (EC 2.3.1.30),    -   7) overexpressed polynucleotide which codes for one or more        components of the glycine cleavage system GcvTHP-Lpd (EC        2.1.2.10, EC 1.4.4.2, EC 1.8.1.4),    -   8) overexpressed polynucleotide which codes for a lipoyl        synthase LipA (EC 2.8.1.8),    -   9) overexpressed polynucleotide which codes for a lipoyl protein        ligase LipB (EC 2.3.1.181),    -   10) overexpressed polynucleotide which codes for a        phosphoglycerate dehydrogenase SerA (EC 1.1.1.95),    -   11) overexpressed polymacleotide which codes for a        3-phosphoserine phosphatase SerB (EC 3.1.3.3),    -   12) overexpressed polynucleotide which codes for a        3-phosphoserine/phosphohydroxythreonine aminotransferase SerC        (EC 2.6.1,52),    -   13) overexpressed polynucleotide which codes for a serine        hydroxymethyltransferase GlyA (EC 2.1.2.1),    -   14) overexpressed polynucleotide which codes for an        aspartokinase I and homoserine dehydrogenase I ThrA (EC 2.7.2.4,        EC 1.1.1.3),    -   15) overexpressed polynucleotide which codes for an aspartate        kinase LysC (EC 2.7.2.4),    -   16) overexpressed polynucleotide which codes for a homoserine        dehydrogenase Hom (EC 1.1.1.3),    -   17) overexpressed polynucleotide which codes for a homoserine        O-acetyltransferase MetX (EC 2.3.1.31),    -   18) overexpressed polynucleotide which codes for a homoserine        O-succinyltransferase MetA (EC 2.3.1.46),    -   19) overexpressed polynucleotide which codes for a cystathionine        gamma-synthase MetE (EC 2.3.1.43),    -   20) overexpressed polynucleotide which codes for a β-C-S-lyase        AecD (EC 4.4.1.8, also called beta-lyase),    -   21) overexpressed polynucleotide which codes for a cystathionine        beta-lyase MetC (EC 4.4.1.8),    -   22) overexpressed polynucleotide which codes for a        B12-independent homocysteine S-methyltransferase MetE (EC        2.1.1.14),    -   23) overexpressed polynucleotide which codes for a B12-dependent        homocysteine S-methyltransferase MetH (EC 2.1.1.13),    -   24) overexpressed polynucleotide which codes for a        methylenetetrahydrofoiate reductase MetE (EC 1.5.1.20),    -   25) overexpressed polynucleotide which codes for one or more        components of the L-methionine exporter BrnFE from        Corynebacteriurn glutamicum,    -   26) overexpressed polynucleotide which codes for one or more        components of the valine exporter YgaZH from Escherichia coli        (b2682, b2683),    -   27) overexpressed polynucleotide which codes for the putative        transporter YjeH from Escherichia coli (b4141),    -   28) overexpressed polynucleotide which codes for one or more        components of the pyridine nucleotide transhydrogenase PntAB (EC        1.6.1.2),    -   29) overexpressed polynucleotide which codes for an        O-succinylhomoserine sulfhydrylase MetZ (EC 2.5.1.48),

30) overexpressed polynucleotide which codes for a phosphoenolpyruvatecarboxylase Pyc (EC 4.1.1.31).

Preferred features here are one or more chosen from the group:

-   -   1) overexpressed polynucleotide which codes for one or more        components of the thiosulphate/sulphate transport system CysPUWA        (EC 3.6.3.25),    -   2) overexpressed polynucleotide which codes for a        3′-phosphoadenosine 5′-phosphosulphate reductase CysH (EC        1.8.4.8),    -   3) overexpressed polynucleotide which codes for one or more        components of the sulphite reductase CysJI (EC 1.8.1.2),    -   4) overexpressed polynucleotide which codes for a cysteine        synthase A CysK (EC 2.5.1.47),    -   5) overexpressed polynucleotide which codes for a. cysteine        synthase B CysM (EC 2.5.1.47),    -   6) overexpressed polynucleotide which codes for a serine        acetyltransferase CysE (EC 2.3.1.30),    -   7) overexpressed polynucleotide which codes for one or more        components of the glycine cleavage system GcvTHP-Lpd (EC        2.1.2.10, EC 1.4.4.2, EC 1.8.1.4),    -   8) overexpressed polynucleotide which codes for a lipoyl        synthase LipA (EC 2.8.1.8),    -   9) overexpressed polynucleotide which codes for a lipoyl protein        lipase LipB (EC 2.3.1.181),    -   10) overexpressed polynucleotide which codes for a        phosphoglycerate dehydrogenase SerA (EC 1.1.1.95),    -   11) overexpressed polynucleotide which codes for a        3-phosphoserine phosphatase SerB (EC 3.1.3.3),    -   12) overexpressed polynucleotide which codes for a        3-phosphoserine/phosphohydroxythreonine aminotransferase SerC        (EC 2.6.1.52),    -   13) overexpressed polynucleotide which codes for a serine        hydroxymethyltransferase GlyA (EC 2.1.2.1),    -   14) overexpressed polynucleotide which codes for an        aspartokinase I and homoserine dehydrogenase I ThrA (EC 2.7.2.4,        EC 1.1.1.3),    -   15) overexpressed polynucleotide which codes for an aspartate        kinase LysC (EC 2.7.2.4),    -   16) overexpressed polynucleotide which codes for a homoserine        dehydrogenase Horn (EC 1.1.1.3),    -   17) overexpressed polynucleotide which codes for a homoserine        acetyltransferase MetX (EC 2.3.1.31),    -   18) overexpressed polynucleotide which codes for a homoserine        O-transsuccinylase MetA (EC 2.3.1.46),    -   19) overexpressed polynucleotide which codes for a cystathionine        gamma-synthase MetB (EC 2.5.1.48),    -   20) overexpressed polynucleotide which codes for a β-C-S-lyase        AecD (EC 4.4.1.8, also called beta-lyase),    -   21) overexpressed polynucleotide which codes for a cystathionine        beta-lyase MetC (EC 4.4.1.8),    -   22) overexpressed polynucleotide which codes tor a        B12-independent homocysteine S-methyltransferase MetE (EC        2.1.1.14),    -   23) overexpressed polynucleotide which codes for a B12-dependent        homocysteine S-methyltransferase MetH (EC 2.1.1.13),    -   24) overexpressed polynucleotide which codes for a        methylenetetrahydrofelate reductase MetF (EC 1.5.1.20).        Very particularly preferred features here are chosen from the        group;    -   1) overexpressed polynucleotide which codes for an aspartokinase        I and homoserine dehydrogenase I ThrA (EC 2.7.2,4, EC 1.1.1.3),    -   2) overexpressed polynucleotide which codes for an aspartate        kinase LysC (EC 2.7,2,4),    -   3) overexpressed polynucleotide which codes for a homoserine        dehydrogenase Hom (EC 1.1.1.3);    -   4) overexpressed polynucleotide which codes for a homoserine        acetyltransferase MetX (EC 2.3.1.31),    -   5) overexpressed polynucleotide which codes for a homoserine        O-transsuccinylase Met A (EC 2.3.1.46),    -   6) overexpressed polynucleotide which codes for a cystathionine        gamma-synthase MetB (EC 2.5.1.48),    -   7) overexpressed polynucleotide which codes for a β-C-S-lyase        AecD (EC 4.4.1.8, also called beta-lyase),    -   8) overexpressed polynucleotide which codes for a cystathionine        beta-lyase MetC (EC 4.4.1.8),    -   9) overexpressed polynucleotide which codes for a        B12-independent homocysteine S-methyltransferase MetE (EC        2.1.1.14),    -   10) overexpressed polynucleotide which codes for a B12-dependent        homocysteine S-methyltransferase MetH (EC 2.1.1.13),    -   11) overexpressed polynucleotide which codes for a        methylenetetrahydrofolate reductase MetF (EC 1.5.1.20).

To improve the production of L-methionine in C. glutamicum, it may beexpedient to attenuate one or more genes chosen from the group of:

-   -   a) a gene pgi coding for glucose 6-phosphate isomerase (Pgi, EC        no. 5.3.1.9),    -   b) a gene thrB coding for homoserine kinase (ThrB, EC no,        2.7.1.39),    -   c) a gene metK coding for S-adenosylmethionine synthase (MetK,        EC no. 2.5.1.6),    -   d) a gene dapA coding for dihydrodipicolinate synthase (DapA, EC        no. 4.2.1.52),    -   e) a gene pck coding for phosphoenolpyruvate carboxykinase (Pck,        EC no. 4.1.1.49),    -   f) a gene cg3086 coding for cystathionine γ-lyase (Cg3086, EC        no. 4.4.1.1),    -   g) a gene cg2344 coding for cystathionine β-synthase (Cg2344, EC        no, 4.2.1.22),    -   h) a gene cg3031 coding for the regulator protein Cg3031,    -   i) a gene mcbR coding for the transcription regulator of        L-methionine biosynthesis (McbR),    -   j) a gene metQ coding for a subunit of the L-methionine        transporter (MetQNI),    -   k) a gene metN coding for a subunit of the L-methionine        transporter (MetQNI),    -   l) a gene metI coding for a subunit of the L-methionine        transporter (MetQNI),    -   m) a gene metP coding for the L-methionine transporter (MetP).

To improve the production of L-methionine in E. coli, it may beexpedient to attenuate one or more genes chosen from the group of:

a) a gene metJ (b3938, ECK3930) coding for the transcription regulatorof L-methionine biosynthesis (MetJ),b) a gene pgi (b4025, ECK4017) coding for glucose 6-phosphate isomerase(Pgi, EC no. 5.3.1.9),c) a gene thrB (b0003, ECK0003) coding for homoserine kinase (ThrB, ECno. 2/7.1.39),d) a gene metK (b2942, ECK2937) coding for S-adenosylmethionine synthase(MetK, EC no. 2,5,1,6),e) a gene dapA (b247 8, ECK24 74) coding for dihydrodipicolinatesynthase (DapA, EC no. 4.2.1,52),f) a gene pck (b3403, ECK3390) coding for phosphoenolpyruvatecarboxykinase (Pck, EC no. 4.1.1.49),g) a gene purU (b1232, ECK1227) coding for formyltetrahydrofolatehydrolase (PurU, EC no. 3.5.1,10),h) a gene pykA (b1854, ECK1855) coding for pyruvate kinase II (PykA, ECno, 2.7.1.40),i) a gene pykF (b1676, ECK1672) coding for pyruvate kinase I (PykF, ECno. 2.7.1.40),j) a gene metQ (b0197, ECK0197) coding for a subunit of the L-methioninetransporter (MetQNI),k) a gene metI (b0198, ECK0198) coding for a subunit of the L-methioninetransporter (MetQNI),l) a gene metN (b0199, ECK0199) coding for a subunit of the L-methioninetransporter (MetQNI),m) a gene dcd (b2065, ECK2059) coding for deoxycytidine 5′-triphosphatedeaminase (Dcd, EC no. 3.5.4.13),n) a gene yncA (b1448, ECK1442) coding for putative N-acyltransferase(YncA, Metabolic Explorer WO2010/020681),o) a gene fnrS (b4699, ECK4511) coding for the regulatory sRNA FnrS.

Brief Description of the Drawing

FIG. 1 schematically shows the pMA-RDL2 plasmid. AmpR: Ampicillinresistance gene; codes for beta-lactamase, upstream: SEQ ID NO: 6;contains recognition sequences for the restriction enzymes Pad and FseIand a ribosome binding site, RBL2: Sequence of the gene RDL2 fromSaccharomyces cerevisiae S288c, downstream; SEQ ID NO; 7; contains asecond stop codon TAA followed by the T1 terminator of the rnpB genefrom E. coli MG1655. Recognition sequences for the restriction enzymesPmeI and SbfI follow after this.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a process for the fermentative preparation ofsulphur-containing amino acids chosen from the group of L-methionine,L-cysteine, L-cystine, L-homocysteine and L-homocystine. The process iscarried out with a microorganism of the family Enterobacteriaceae orwith a microorganism of the family Corynebacteriaceae whichoverexpresses a gene coding for a polypeptide with the enzymaticactivity of a thiosulphate sulphurtransferase,

The term “gene” here means a section on the deoxyribonucleic acid (DNA)which contains the information for the production (transcription) firstof a ribonucleic acid (RNA), and this the Information for the production(translation) of a protein (polypeptide), here a polypeptide with theactivity of a thiosulphate sulphurtransferase. The fact that a gene or apolynucleotide contains the information for production of a protein isalso called coding of a protein or polypeptide by the gene or by theRNA. Endogenous genes or polynucleotides are understood as meaning theopen reading frames (ORF), genes or alleles or polynucleotides thereofpresent in the population of a species. The terms “gene” and “ORF” (openreading frame) are used synonymously in this invention.

The term “polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, which can be non-modified RNA or DNA ormodified RNA or DNA.

The term “polypeptide” indicates peptides or proteins which contain twoor more amino acids joined via peptide bonds. The terms polypeptide andprotein are used as synonyms. Proteins belong to the base units of allcells. They not only impart structure to the cell, but are the molecular“machines” which transport substances, catalyse chemical reactions andrecognize signal substances.

“Proteinogenic amino acids” are understood as meaning the amino acidswhich occur in natural proteins, that is to say in proteins ofmicroorganisms, plants, animals and humans. These include, inparticular, L-amino acids chosen from the group of L-aspartic acid,L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine,glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan, L-proline and L-arginine, as well as selenocysteine. Inthis context, the proteinogenic amino acids are always α-amino acids.Apart from the amino acid glycine, for all proteinogenic amino acids theα-carbon atom is asymmetric (the molecules are chiral): Two enantiomersof each of these amino acids exist. In this context, only one of the twoenantiomers is proteinogenic, and in fact the L-amino acid: theapparatus necessary for building up the proteins—the ribosome, the tRNA,the aminoacyl-tRNA synthetase (this loads the tRNA with amino acids) andothers—are themselves also chiral and can recognize only the L-variants.

The term “gene expression” (“expression” for short) in general indicatesthe expression, of the genetic information to form a phenotype. In thenarrower sense, gene expression indicates the transcription of a gene toan RNA and the subsequent translation of the RNA to a polypeptide, whichmay have an enzymatic activity.

The term “overexpression” is understood generally as meaning an increasein the intracellular concentration or activity of a ribonucleic acid, aprotein or an enzyme compared with the starting strain (parent strain)or wild-type strain. In the case of the present invention, thiosulphatesulphurtransferase genes or polynucleotides which code for athiosulphate sulphurtransferase polypeptide are overexpressed.

A “starting strain” (parent strain) is understood as meaning themicroorganism strain, on which measures to increase the productivity ofone or more amino acids, peptides or proteins, or measures to increasethe activity of one or more enzymes (e.g. a measure leading tooverexpression) are performed. A starting strain can be a wild-typestrain, but also a strain which has already been modified previously(for example a production strain).

A “wild type” of a ceil preferably indicates a cell whose genome is in astate such as is formed naturally by evolution. The term is used bothfor the entire cell and for individual genes. Thus in particular thosecells or those genes whose gene sequences have been at least partlymodified by humans by means of recombinant methods do not fall under theterm “wild type”.

The mutants obtained in the context of this invention show an increasedsecretion or production of the desired amino acid in a fermentationprocess compared with the starting strain or parent strain employed. Inthis context, the amino acids are released, into the medium surroundingthem or are accumulated inside the cell (accumulation).

The term “increase” or “increased activity” in this connection describesthe increase in the intracellular enzymatic activity of one or moreenzymes in a microorganism, which are coded by the corresponding DNA.

In principle, an increase in the enzymatic activity can be achieved, forexample, by increasing the number of copies of the gene sequence or genesequences which code for the enzyme, using a potent promoter or using agene or allele which codes for a corresponding enzyme with an increasedactivity, and optionally combining these measures. Cells which have beengenetically modified according to the invention are produced, forexample, by transformation, transduction, conjugation, or a combinationof these methods with a vector which contains the desired gene, anallele of this gene or parts thereof and a vector which renders possibleexpression of the gene. Heterologous expression is achieved inparticular by integration of the gene or the alleles into the chromosomeof the cell or an extrachromosomally replicating vector.

An overview of the possibilities of increasing the enzymatic activity incells by the example of pyruvate carboxylase is given in DE-A-100 31 939which is incorporated herewith by reference and the disclosure contentof which with respect to the possibilities for increasing the enzymaticactivity in cells forms a part of the disclosure of the presentinvention.

The increase in enzymatic activity can be achieved, for example, byincreasing the number of copies of the corresponding polynucleotideschromosomally or extrachromosomally by at least one copy.

A widely used method for increasing the number of copies comprisesincorporating the corresponding polynucleotide into a vector, preferablya plasmid, which is replicated by a bacterium.

Suitable plasmid vectors for Enterobacteriaceae are e.g. cloning vectorsderived from pACYC184 (Bartolomé et al.; Gene 102: 75-73 (1991)),pTrc99A (Amann et al.; Gene 69: 301-315 (1988)) or pSC101 derivatives(Vocke and Bastia; Proceedings of the National Academy of Sciences USA80(21): 6557-6561 (1983)). Plasmids derived from pCL1920 (Lerner, C. G.and Inouye, M., Nucl. Acids Res. (1990)18:4631 [PMID: 2201955]) arefurthermore particularly suitable. Plasmid vectors derived frombacterial artificial chromosomes (BAG), such as e.g. pCC1BAC (EPICENTREBiotechnologies, Madison, USA), are likewise suitable for increasing thenumber of copies of the corresponding polynucleotides in E. coli.

Suitable plasmid vectors for C. glutamicum are, for example, pZ1 (Menkelet al., Applied and Environmental Microbiology 64: 549-554 (1989),pEKEx1 (Eikmanns et. al., Gene 107: 69-74 (1991)) or pHS2-l (Sonnen etal., Gene 107:69-74 (1991)) . They are based on the cryptic plasmidspHM1519, pBL1 or pGA1. Other plasmid vectors, such as, for example,those which are based on pCG4 (U.S. Pat. No. 4,489,160) or pNG2(Serwold-Davis et al., FEMS Microbiology Letters 66: 119-124 (1990)) orpAG1 (U.S. Pat. No. 5,158,891), can be employed in the same manner. Anoverview article on the subject of plasmids in C. glutamicum is to befound in Tauch et al. (Journal of Biotechnology 104, 27-40 (2003)).

Transposons, insertion elements (IS elements) or phages can furthermorebe employed as vectors. Such genetic systems are described, for example,in the patent specifications U.S. Pat. No. 4,822,738, U.S. Pat. No.5,804,414 and U.S. Pat. No. 5,804,414. The IS element ISaB1 described inWO 92/02627 or the transposon Tn 45 of the plasmid pXZ10142 (referred toin “Handbook of Corynebacterium glutamicum” (editors; L. Eggeling and M.Bott)) can be used in the same manner,

Another widespread method for achieving overexpression is the method ofchromosomal gene amplification. In this method, at. least one additionalcopy of the polynucleotide of interest, is inserted into the chromosomeof a bacterium. Such amplification methods are described, for example,in WO 03/014330 or WO 03/040373.

A further method for achieving overexpression comprises linking thecorresponding gene or allele in a functional manner (operably linked)with a promoter or an expression cassette. Suitable promoters for C.glutamicum are described, for example, in FIG. 1 of the overview articleby Patek et al. (Journal of Biotechnology 104(1-3), 311-323 (2003)). Thevariants of the dapA promoter, for example the promoter A25, described,by Vasicova et al, (Journal of Bacteriology 181, 6188-6191 (1999)), canbe employed in the same manner. The gap promoter of C. glutamicum (EP06007373) can furthermore be used.

For E. coli e.g. the promoters T3, T7, SP6, M13, lac, tac and trcdescribed by Amann et al. (Gene 69(2), 301-315 (1988)) and Amann andBrosius (Gene 40(2-3), 183-190(1985)) are known, some of which can alsobe used for C. glutamicum. Such a promoter can be inserted, for example,upstream of the gene in question, typically at a distance ofapproximately 1-500 nucleobases from, the start codon. U.S. Pat. No.5,939,307 reports that by incorporation of expression cassettes orpromoters, such as, for example, the tac promoter, trp promoter, lpppromoter or PL promoter and PR promoter of the phage λ, for exampleupstream of the chromosomal threonine operon, it was possible to achievean increase in the expression. The promoters of phage T7, the gear-boxpromoter or the nar promoter can be used in the same manner. Suchexpression cassettes or promoters can also be used to overexpressplasmid-bound genes, as described in EP 0 593 792. By using the lacIQallele, expression of plasmid-bound genes can in turn be controlled(Glascock and Weickert, Gene 223, 221-231 (1998)). It is furthermorepossible for the activity of the promoters to be increased, bymodification of their sequence by means of one or more nucleotideexchanges, by insertion(s) and/or deletion(s).

By the measures of overexpression, the activity or concentration of thecorresponding polypeptide is in general increased by at least 10%, 25%,50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, by a maximum of up to1,000% or 2,000%, based on the activity or concentration of thepolypeptide in the corresponding starting strain (wild type or startingmicroorganism) before the measure leading to overexpression.

Methods for determination of the enzymatic activity of thiosulphatesulphurtransferases are described, for example, by Cheng H, Donahue J L,Battle S E, Ray W K, Larson T J, 2008, Open Microbiol J., 2: 18-28 andby Alexander K., Volini M, 1987, J. Biol. Chem., 262: 6595-6604.

The expression of the abovementioned enzymes or genes can be detectedwith the aid of 1- and 2-dimensional protein gel separation andsubsequent optical identification of the protein concentration in thegel with appropriate evaluation software. If the increase in an enzymeactivity is based exclusively on an increase in the expression of thecorresponding gene, the increase in the enzyme activity can bequantified in a simple manner by a comparison of the 1- or 2-dimensionalprotein separations between the wild type and the genetically modifiedcell, A conventional method for preparation of the protein gels in thecase of bacteria and for identification of the proteins is the proceduredescribed by Hermann et al. (Electrophoresis; 22: 1712.23 (2001)). Theprotein concentration can likewise be analysed by western blothybridization with an antibody specific for the protein to be detected(Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989) andsubsequent optical evaluation with appropriate software fordetermination of the concentration (Lohaus and Meyer (1989) Biospektrum,5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647). Theactivity of DNA-binding proteins can be measured by means of DNA bandshift assays (also called gel retardation; Wilson et al. (2001) Journalof Bacteriology, 183: 2151-2155). The action of DNA-binding proteins onthe expression of other genes can be detected by various well-describedmethods of reporter gene assay (Sambrook et al., Molecular Cloning: alaboratory manual, 2nd ed. Cold Spring harbor Laboratory Press, ColdSpring Harbor, N.Y. USA, 1989). The intracellular enzymatic activitiescan be determined by various methods which have been described (Donahueet al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al,(2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al.(1973) Journal of Bacteriology 115 (3): 816-823) . If no concretemethods for determination of the activity of a particular enzyme aregiven in the following, the determination of the increase in enzymeactivity and also the determination of the reduction in an enzymeactivity are preferably carried out by means of the methods described inHermann et al., Electophoresis, 22; 1712-23 (2001), Lohaus et al.,Biospektrum 5 32-39 (1393), Lottspeich, Angewandte Chemie 111: 2630-2647(1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001).

If the increase in enzyme activity is effected by mutation of theendogenous gene, such mutations can be generated either by conventionalmethods in a non-targeted manner, such as, for example, by UVirradiation or by mutation-inducing chemicals, or in a targeted mannerby means of genetic engineering methods, such as deletion(s),insertion(s) and/or nucleotide exchange(s). Genetically modified cellsare obtained by these mutations. Particularly preferred mutants ofenzymes are in particular also those enzymes which can no longer befeedback-inhibited, or at least can be feedback-inhibited to a lesserextent compared with the wild-type enzyme.

If the increase in enzyme activity is effected by increasing theexpression of an enzyme, the number of copies, for example, of thecorresponding genes is increased or the promoter and regulation regionor the ribosome binding site upstream of the structural gene aremutated. Expression cassettes which are incorporated upstream of thestructural gene act in the same manner. By inducible promoters it isadditionally possible to increase the expression at any desired point intime. Furthermore, however, so-called enhancers can be assigned to theenzyme gene as regulatory sequences, which likewise have the effect ofan increased gene expression via an improved interaction between the RNApolymerase and DNA, The expression is likewise improved by measures forprolonging the life of the mRNA, The enzyme activity is furthermorelikewise increased by preventing degradation of the enzyme protein. Inthis context, the genes or gene constructs are either present inplasmids with a different number of copies, or are integrated in thechromosome and amplified. Alternatively, an overexpression of the genesin question can furthermore be achieved by modifying the mediacomposition and culture procedure. Instructions on this are to be foundby the person skilled in the art inter alia in Martin et al.(Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-43C (1988)), inEikmanns et al. (Gene 102, 93-98 (1991)), in EP-A-G 472 869, in U.S.Pat. No. 4,601,833, in Schwarzer and Pühler (Bio/Technology 9, 84-87(1991)), in Reinscheid et al. (Applied and Environmental Microbiology60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175,1001-1007 (1993)), in WO-A-96/15246, in Malumbres et al. (Gene 134,15-24 (1993)), in JP-A-10-229831, in Jensen and Hammer (Biotechnologyand Bioengineering 58, 191-195 (1998)) and in known textbooks ofgenetics and molecular biology. The measures described above, like themutations, lead to genetically modified cells.

Those plasmid vectors with the aid of which the method of geneamplification by integration into the chromosome can be used arefurthermore also suitable, such as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60: 126-132(1994)) for duplication or amplification of the hom-thrB operon. In thismethod, the complete gene is cloned into a plasmid vector which can bereplicated in a host (typically Escherichia coli), but not inCorynebacterium glutamicum. Possible vectors are, for example, pSUP301(Simon et al., Bio/Technology 1: 784-791 (1983)), pK18mob or pK19mob(Schafer et al., Gene 145: 59-73 (1994)), pGEM-T (Promega Corporation,Madison, Wis., USA), pCR2.1-TOPO (Shuman, Journal of BiologicalChemistry 269: 32678-84 (1394)), pCR-BluntII-TOPO (Invitrogen,Groningen, The Netherlands), pEM1 (Schrumpf et al., Journal ofBacteriology 173:4510-4516)) or pBGS8 (Spratt et al., Gene 41: 337-342(1986)), The plasmid vector which contains the gene to be amplified isthen converted into the desired strain of Corynebacterium glutamicum byconjugation or transformation. The method of conjugation is described,for example, by Schäfer et al., Applied and Environmental Microbiology60: 756-759 (1994). Methods for transformation are described, forexample, by Thierbach et al., Applied Microbiology and Biotechnology 29:356-362 (1938), Dunican and Shivnan, Bio/Technology 7: 1067-1070 (1989)and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). Afterhomologous recombination by means of a cross-over event, the resultingstrain contains at least two copies of the gene in question. A similarmethod for E. coli is described, for example, by Link, A. J., Phillips,D. and Church, G. M. (1997), J. Bacteriology 179: 6228-6237.

For insertion or deletion of DNA in the chromosome, recombinase-mediatedmethods can also be used, such as have been described, for example, byDatsenko K A, Wanner B L., 2000, Proc Natl Acad Sci USA., 97(12):6640-5.

The wording “an activity of an enzyme which is increased compared withits wild-type strain or starring strain” used above and in the followingis preferably always to be understood, as meaning an activity of theparticular enzyme which has been increased by a factor of at least 2,particularly preferably of at least 10, moreover preferably of at least100, moreover still more preferably of at least 1,000 and mostpreferably of at least 10,000. The cell according to the invention whichhas “an activity of an enzyme which is increased compared with itswild-type strain or starting strain” furthermore also includes inparticular a cell whose wild type or starting strain has no or at leastno detectable activity of this enzyme and which shows a detectableactivity of this enzyme only after increasing the enzyme activity, forexample by overexpression, In this connection, the term “overexpression”or the wording “increase in expression” used in the following alsoincludes the case where a starting cell, for example a wild-type cell,has no or at least no detectable expression and a detectable expressionof the enzyme is induced only by recombinant methods.

The isolated bacteria obtained by the measures of the invention show anincreased secretion or production of the desired amino acid in afermentation process compared with the starting strain or parent strainemployed.

Isolated bacteria are to be understood as meaning the mutants andrecombinant bacteria according to the invention which have been isolatedor produced, in particular of the Enterobacteriaceae orCorynebacteriaceae family, and which have an increased activity of athiosulphate sulphurtransferase compared with the starting strain.

The output of the bacteria isolated and of the fermentation processusing the same with, respect to one or more of the parameters chosenfrom the group of product concentration (product per volume), productyield (product formed per carbon source consumed) and product, formation(product, formed per volume and time), or also other process parametersand combinations thereof, is improved by at least 0.5%, at least 1%, atleast. 1.5% or at least 2%, based on the starting strain or parentstrain or the fermentation process using the same.

In the process according to the invention, the bacteria can becultivated continuously—as described, for example, inPCT/EP2004/008882—or discontinuously in the batch process (batchcultivation) or in the fed batch (feed process) or repeated fed batchprocess (repetitive feed process) for the purpose of production ofL-amino acids. A summary of a general nature of known cultivationmethods is available in. the textbook by Chmiel (Bioprozesstechnik l.Einführung in die Bioverfahrenstechnik (Gustav Fischer Veriag,Stuttgart, 1991) ) or in the textbook, by Storhas (Bioreaktoren undperipnere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium or fermentation medium to be used must meet therequirements of the particular strains in a suitable manner.Descriptions of culture media for various microorganisms are containedin the handbook “Manual of Methods for General Bacteriology” of theAmerican Society for Bacteriology (Washington D.C., USA, 1981) . Theterms culture medium and fermentation medium or medium, are mutuallyinterchangeable.

Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, sucrose-containing solutions from sugarbeet or cane sugar production, starch, starch hydrolysate and cellulose,oils and fats, such as, for example, soya oil, sunflower oil, groundnutoil and coconut fat, fatty acids, such as, for example, palmitic acid,stearic acid and linoleic acid, alcohols, such as, for example,glycerol, methanol and ethanol, and organic acids, such as, for example,acetic acid, can be used as the source of carbon. These substances canbe used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya bean flour and urea,or inorganic compounds, such as ammonium sulphate, ammonium chloride,ammonium phosphate, ammonium, carbonate and ammonium nitrate, can beused as the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus.

According to the invention, the source of sulphur employed is a salt ofdithiosulphuric acid (thiosulphate), optionally together with othersources of sulphur, such as, for example, sulphate, sulphite ordithionite.

The culture medium must furthermore contain salts, for example in theform of chlorides, of metals, such as, for example, sodium, potassium,magnesium, calcium and iron, such as, for example, magnesium sulphate oriron sulphate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids, for example homoserine, and vitamins,for example thiamine, biotin or pantothenic acid, can be employed inaddition to the abovementioned substances.

Suitable precursors of the particular amino acid, can moreover be addedto the culture medium.

The starting substances mentioned can be added to the culture in theform, of a single batch, or can be fed in during the cultivation in asuitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulphuric acid, can be employed in a suitable manner to control the pHof the culture. The pH is in general adjusted, to a value of from 6.0 to9.0, preferably 6.5 to 8. Antifoams, such as, for example, fatty acidpolyglycol esters, can be employed to control the development of foam.Suitable substances having a selective action, such as, for example,antibiotics, can be added to the medium to maintain the stability ofplasmids. To maintain aerobic conditions, oxygen or oxygen-containinggas mixtures, such as, for example, air, are introduced into theculture. The use of liquids which are enriched with hydrogen peroxide islikewise possible. The fermentation is optionally carried out underincreased pressure, for example under a pressure of from 0.03 to 0.2MPa, The temperature of the culture is usually 20° C. to 45° C., andpreferably 25° C. to 40° C. In the batch process the cultivation iscontinued until a maximum of the desired amino acid has formed. Thistarget is usually reached within 10 hours to 160 hours. In continuousprocesses longer cultivation times are possible.

Suitable fermentation media are described, inter alia, in U.S. Pat. No.6,221,636, in U.S. Pat. No. 5,840,551, in U.S. Pat. No. 5,770,409, inU.S. Pat. No. 5,605,818, in U.S. Pat. No. 5,275,940 and in U.S. Pat. No.4,224,409,

Methods for the determination of L-amino acids are known from the priorart. The analysis can be carried out, for example, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190) by ion exchangechromatography with subsequent ninhydrin derivatization, or it can becarried out by reversed phase HPLC, as described by Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

The fermentation broth produced in this manner is then further processedto a solid or liquid product.

A fermentation broth is understood as meaning a fermentation medium inwhich a microorganism has been cultivated for a certain time and at acertain temperature. The fermentation medium and/or the medium employedduring the fermentation contain(s) ail the substances and componentswhich ensure a multiplication of the microorganism and a formation ofthe desired amino acid.

At the conclusion of the fermentation of the fermentation broth formedaccordingly contains a) the biomass of the microorganism formed as aresult of the multiplication of the ceils of the microorganism, b) thedesired amino acid, formed in the course of the fermentation, c) theorganic by-products formed in the course of the fermentation and d) theconstituents of the fermentation medium/fermentation, media employed orof the starting substances which have not been consumed by thefermentation, such as, for example, vitamins, such as biotin, aminoacids, such as homoserine, or salts, such as magnesium sulphate.

The organic by-products include substances which are possibly produced,in addition to the particular desired L-amino acid, and possiblysecreted by the microorganisms employed in the fermentation. Theseinclude L-amino acids which make up less than 30%, 20% or 10%, comparedwith, the desired amino acid. They furthermore include organic acidswhich carry one to three carboxyl groups, such as, for example, aceticacid, lactic acid, citric acid, malic acid or fumaric acid. Finally,they also include sugars, such as, for example, trehalose.

Typical fermentation broths which are suitable for industrial purposeshave an amino acid content of from 40 g/kg to 130 g/kg or 50 g/kg to 150g/kg. The content of biomass (as dried biomass) is in general 20 to 50g/kg.

EXAMPLES Example 1 Synthesis and Cloning of the Gene RDL2

For the expression of the ORF RDL2 (SEQ ID NO: 1) from S. cerevisiae, anucleotide sequence (GEINIEART AG; Regensburg, Germany) which comprisesan upstream sequence (“upstream”, SEQ ID NO: 6), the sequence of ORFRDL2 (“RDL2”, SEQ ID NO: 1) and a downstream sequence (“downstream”, SEQID NO: 7) was synthesized da novo. The upstream sequence containsrecognition sequences for the restriction enzymes PacI and FseI and aribosome binding site. The downstream sequence contains a second stopcodon TAA followed by the T1 terminator of the rnpB gene from E. coliMG1655. Recognition sequences for the restriction enzymes PmeI and SbfIfollow after this. After the synthesis, the nucleotide sequence wascleaved with SacI and KpnI and cloned into the plasmid pMA (ampR),likewise cleaved with SacI and KpnI, The resulting plasmid wasdesignated “pMA-RDL2” (SEQ ID NO: 8, FIG. 1).

Starting from the amino acid sequence of the protein Rd12p coded by theORF RDL2 (sequence “RDL2p”, SEQ ID NO: 2), three different DNA sequenceswhich code for Rd12p proteins with the wild-type amino acid sequencewere generated. The sequences were designated “RDL2a” (SEQ ID NO: 3),“RDL2b” (SEQ ID NO: 4) and “RDL2c” (SEQ ID NO: 5). These three sequenceswere each synthesized de novo together with the upstream and downstreamsequences described above and cloned into the plasmid pMA as describedabove (GENEART AG; Regensburg, Germany). The resulting plasmids weredesignated pMA-RDL2a, pMA-RDL2b and pMA-RDL2c. The variants mentioneddiffer in the optimization of the codon usage of the microorganism usedfor the process. The values for the adaptation to the codon usage arethus as follows:

RDL2: Codon usage not adapted (codon adaptation index CAI=0.27)RDL2a: Codon usage slightly adapted to E. coli (CAI=0.38)RDL2b: Codon usage more highly adapted to E. coli (CAI=0.72)RDL2c: Codon usage completely adapted, to E. coli (CAI - 1)

Examples 2 Cloning of the QRFs RDL2, RDL2a, RDL2b and RDL2c into thePlasmid pME101-thrA*1-cysE-Pgap-matA*11

For the expression, in E. coli, the four gene variants coding for theRDL2 protein were each cloned into the E. coli production plasmidpME101-thrA*1-cysE-Pgap-metA*11 (WO2007/077041) downstream of metA*11.

For this, the genes were each amplified with, the primers RDL2-t4-f (SEQID NO: 11) and RDL2-r (SEQ ID NO: 12) using the polymerase chainreaction (PCR) with Phusion DNA Polymerase (Finnzymes Oy, Espoo,Finland). The plasmids PMA-RDL2, pMA-RDL2a, pMA-RDL2b and pMA-RDL2c fromExample 1 served as templates.

RDL2-t4-f (SEQ ID NO: 11): 5′aggacagtcgacggtaccgcaagcttcggcttcgcACTGGAAAGCGG GCAGTGAG 3′RDL2-r (SEQ ID NO: 12): 5′ AGCGCGACGTAATACGACTC 3′

The PGR products 804 bp in size and the plasmidpME101-thrA*1-cysE-PgapA-metA*11 were cleaved with the restrictionenzymes SalI and SbfI, ligated and transformed into the E. coli strainDK5α. Plasmid-carrying cells were selected by cultivation on LB agarwith 50 μg/ml of streptomycin. After isolation of the plasmid DNA,successful cloning was detected, by control cleavage with therestriction enzyme AgeI. Finally, the cloned DNA fragments weresequenced with the following primers:

pME-RDL2-Seqf (SEQ ID NO: 13): 5′ ATGTGGAAGCCGGACTAGAC 3′pME-RDL2-Seqr (SEQ ID NO: 14): 5′ TCGGATTATCCCGTGACAGG 3′

The plasmids formed in this way were designated pMS-RDL2, pME-RDL2a,pME-RDL2b and pME-RDL2c.

Example 3 Transformation of E. coli MG1655ΔmetJ Ptrc-metH Ptrc-metFPtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP

The E. coil strain MG1655ΔmetJ metA*11 Ptrc-metH Ptrc-metFPtrcF-cysPUMAM PtrcF-cysJIH (designated DM2219 in the following) whichproduces L-methionine is described in the parent specification WO2009/043803 A2.

The strain was transformed in each case with the plasmids pME-RDL2,pME-RDL2a, pME-RDL2b and pME-RDL2c and with the plasmidpME101-thrA*1-cysE-PgapA-metA*11 (WO2009/043803 A2) and plasmid-carryingcells were selected on LB agar with 50 μg/ml of streptomycin. Thetransformants were transinoculated into in each case 10 ml of LB liquidmedium with 1% glucose and 50 μg/ml of streptomycin and were cultivatedat 37° C. for 16 hours. Glycerol was then added to a final concentrationof 10% and the cultures were frozen at −70° C.

Example 4 Evaluation of the E. coli L-methionine Production Strains

The output capacity of the E. coli L-methionine production strains wasevaluated by production experiments in 100 ml conical flasks. Asprecultures, in each case 10 ml of preculture medium (10% LB medium with2.5 g/l of glucose and 90% PCI minimal medium.) were inoculated with1.00 μl of cell culture and the cultures were cultivated at 37° C. for10 hours. In each case 10 ml of PC1 minimal medium (Table 1) were theninoculated with these to an OD 600 nm of 0.2 (Eppendorf Bio-Photometer;Eppendorf AG, Hamburg, Germany) and cultivated at 37° C. for 24 hours.The extracellular L-methionine concentration was determined with anamino acid analyzer (Sykam GmbH, Erasing, Germany) by ion exchangechromatography and post-column derivatization with ninhydrin detection.The extracellular glucose concentration was determined with a YSI 2700Select Glucose Analyzer (YSI Life Sciences, Yellow Springs, Ohio, USA).The data show that the expression of ORE RDL2, RDL2a, RDL2b or RDL2csignificantly increases the L-methionine concentration (Table 2).

TABLE 1 PCl minimal medium Substance Concentration ZnSO₄ * 7 H₂O 4 mg/lCuCl₂ * 2 H₂O 2 mg/l MnSO₄ * H₂O 20 mg/l CoCl₂ *6 H₂O 8 mg/l H₃BO₃ 1mg/l Na₂MoO₄ * H₂O 0.4 mg/l MgSO₄ * 7 H₂O 1 g/l Citric acid * 1 H₂O 6.56g/l CaCl₂ * 2 H₂O 40 mg/l K₂HPO₄ 8.02 g/l Na₂HPO₄ 2 g/l (NH₄)₂HPO₄ 8 g/lMOPS 5 g/l NaOH 10M adjusted to pH 6.8 FeSO₄ * 7 H₂O 40 mg/l Thiaminehydrochloride 10 mg/l Vitamin B12 10 mg/l Glucose 10 g/1 Isopropyl thio-β-galactoside 2.4 mg/l (IPTG) Spectinomycin 50 mg/l

TABLE 2 L-Methionine concentrations in the fermentation broths of thevarious plasmid-carrying E. coli DM2219 strains OD L-Methionine StrainPlasmid (600 nm) (g/l) DM2219 pME101-thrA*1-cysE- 7.51 0.43PgapA-metA*11 DM2219 pME-RDL2 7.59 0.51 DM2219 pME-RDL2a 7.72 0.53DM2219 pME-RDL2b 7.50 0.52 DM2219 pME-RDL2c 7.64 0.54

Example 5 Cloning of the ORFs RDL2, RDL2a, RDL2b and RDL2c into thePlasmid pEC-XT99A

As the base vector for expression of the ORFs RDL2, RDL2a, RDL2b andRDL2c in C. glutamicum, the E. coli-C. glutamicum shuttle expressionplasmid pEC-XT99A (EPI085094B1) was used. The plasmids pMA-RDL2,pMA-RDL2a, pMA-RDL2b and pMA-RDL2c from Example 1 were each cleaved withthe restriction enzymes StuI and NaeI and separated in a 0.8% strengthagarose gel. The DNA fragments 638 bp in size were cut out of the geland the DNA was extracted (QIAquick Gel Extraction Kit, QIAGEN, Hilden,Germany). The expression plasmid pEC-XT99A was cleaved with therestriction enzyme Ecl136II. Ligation was then in each case carried outwith the DNA fragments 638 bp in size using the Ready-To-Go Ligation Kit(Amersham GE Healthcare Europe GmbH, Freiburg, Germany). The E. colistrain DH5α (Invitrogen GmbH; Darmstadt; Germany) was transformed withthe ligation, batches and plasmid-carrying cells were selected on LBagar with 5 μg/ml of tetracycline.

After isolation of the plasmid DNA, successful cloning was detected by acontrol cleavage with the restriction enzymes PmeI and SacII. Finally,the plasmids were sequenced with the following primers:

pECf (SEQ ID NO: 9): 5′ TACTGCCGCCAGGCAAATTC 3′ pECr (SEQ ID NO: 10): 5′TTTGCGCCGACATCATAACG 3′

The plasmid constructs in which the plasmid's own trc promoter and theORFs coding for RDL2 have the same orientation were designated asfollows: pEC-RDL2, pEC-RDL2a, pEC-RDL2b and pEC-RDL2c.

Example 6 Transformation of C. glutamicum M1179

The strain Corynebacteriurn glutamicum M1179 is an ethionine-resistantproducer of L-methionine (WO2007/011939). It was deposited at theDeutsche Sammlung fur Mikroorganismen und Zellkuituren (DSMZ,Braunschweig, Germany) on May 18, 2005 as DSM17322 under the terms ofthe Budapest Treaty.

The plasmids pEC-RDL2, pEC-RDL2a, pEC-RDL2b, pEC-PDL2c and pEC-XT99Awere electroporated into the strain M1179 in accordance with theelectroporation conditions of Tauch et al. (1994, FEMS MicrobiologicalLetters, 123: 343-347).

Selection of plasmid-carrying cells was performed on LB agar with 5μg/ml of tetracycline. The plasmid DNA of the transformants was isolated(Peters-Wendisch et al., 1998, Microbiology 144, 915-927) and checked byrestriction cleavage and gel electrophoresis. The strains formed in thisway were designated M1179/pEC-RDL2, M1179/pEC-PDL2a, M1179/pEC-RDL2b,M1179/pEC-RDL2c and M1179/pEC-XT99A.

Example 7 Evaluation of the C. glutamicum L-methionine ProductionStrains

The C. glutamicum strains M1179/pEC-RDL2 , M1179/pEC-RDL2a,M1179/pEC-RDL2b, M1179/pEC-RDL2c and M1179/pEC-XT99A produced werecultivated in a nutrient medium suitable for the production ofL-methionine and the L-methionine content in the culture supernatant wasdetermined.

For this, the strains were first smeared onto agar plates (brain-heartagar with kanamycin (25 mg/l)) and incubated at 33° C. for 24 hours.From these agar plate cultures, cells were trans inoculated into in eachcase 10 ml of BK medium (Hirn-Kerz Bouillon, Merck, Darmstadt, Germany)with 5 mg/l of tetracycline and the cultures were shaken in 100 mlconical flasks at 33° C. and at 240 rpm for 24 hours. In each case 10 mlof PM medium (Table 3) were then inoculated with these to an OD 560 nmof 0.1 (Genios, Tecan Deutschland GmbH, Crailsheim, Germany) andcultivated at 33° C. in 100 ml conical flasks with baffles for 24 hours.

TABLE 3 PM medium Substance Concentration Glucose 50 g/l (NH₄)₂S₂O₃ 10g/l (NH₄)Cl 10 g/l MgSO₄ * 7 H₂O 0.4 g/l KH₂PO₄ 0.6 g/l Yeast extract(Difco) 10 g/l FeSO₄ * 7 H₂O 2 mg/l MnSO₄ * H₂O 2 mg/l Aqueous ammoniaadjusted to pH 7.8 Thiamine * HCl 1 mg/l Vitamin B12 0.2 mg/l Biotin 0.1mg/l Pyridoxine * HCl (vitamin B6) 5 mg/l Threonine 238 mg/l CaCO₃ 50g/l Tetracycline 5 mg/l Isopropyl thio-β-galactoside (IPTG) 1 mM

After 24 hours the OD at 660 nm was determined (Genios, TecanDeutschland GmbH, Crailsheim, Germany) and the concentration ofL-methionine formed was measured. L-Methionine was determined with anamino acid analyser (Sykam GmbH, Erasing, Germany) by ion exchangechromatography and post-column derivatization with ninhydrin detection.Table 4 shows the optical densities and the L-methionine concentrationsof the cultures. The expression of the ORF RDL2, RDL2a, RDL2b or RDL2cresults in a significant increase in the L-methionine concentration.

TABLE 4 Methionine concentrations in the fermentation broths of thevarious plasmid-carrying C. glutamicum M1179 strains OD L-MethionineStrain (600 nm) (g/1) M1179/pEC-XT99A 25.7 0.47 M1179/pEC-RDL2 25.8 0.50M1179/pEC-RDL2a 24.1 0.50 M1179/pEC-RDL2b 26.2 0.55 M1179/pEC-RDL2c 24.50.54

1. A process for the fermentative preparation of sulphur-containing amino acids chosen from the group of L-methionine, L-cysteine, L-cystine, L-homocysteine and L-homocystine, comprising the steps: a) provision of a microorganism of the family Enterobacteriaceae or of a microorganism of the family Corynebacteriaceae which has an increased thiosulphate sulphurtransferase activity compared with the particular starting strain; b) fermentation of the microorganism from a) in a medium which contains an inorganic source of sulphur from the group of salt of dithiosulphuric acid or a mixture of a salt of dithiosulphuric acid and a salt of sulphuric acid, a fermentation broth being obtained, and c) concentration of the sulphur-containing amino acid in the fermentation broth from b).
 2. A process for the fermentative preparation of sulphur-containing amino acids chosen from the group of L-methionine, L-cysteine, L-cystine, L-homocysteine and L-homocystine, preferably according to claim 1, comprising the steps: a) provision of a microorganism of the family Enterobacteriaceae or of a microorganism of the family Corynebacteriaceae which overexpresses a gene coding for a polypeptide with the activity of a thiosulphate sulphurtransferase; b) fermentation of the microorganism from a) in a medium which contains an inorganic source of sulphur from the group of salt of dithiosulphuric acid or a mixture of a salt of dithiosulphuric acid and a salt of sulphuric acid, a fermentation broth being obtained, and c) concentration of the sulphur-containing amino acid in the fermentation broth from b).
 3. The process according to claim 1, wherein the microorganism overexpresses one or more genes coding for a polypeptide with the activity of a thiosulphate sulphurtransferase, the polypeptide with the activity of a thiosulphate sulphurtransferase selected from the group consisting of: a) a polypeptide consisting of or containing the polypeptides Rdl2p, GlpE, PspE, YgaP, ThiI, YbbB, SseA, YnjE, YceA, YibN, NCgl0671, NCgl1369, NCgl2616, NCgl0053, NCg10054, NCgl2678, NCgl289G; a thiosulphate sulphurtransferase from mammals, for example the thiosulphate sulphurtransferase from the bovine liver (Bos taurus); preferably Rdl2p, GlpE, PspE and particularly preferably Rdl2p; b) a polypeptide consisting of or containing the amino acid sequence shown in SEQ ID NO: 2; c) a polypeptide with an amino acid sequence which is identical to the extent of 70 % or more to the amino acid sequence of a) or b), the polypeptide having thiosulphate sulphurtransferase activity; and d) a polypeptide which has an amino acid sequence containing a deletion, substitution, insertion and/or addition of from 1 to 45 amino acid residues with respect to the amino acid sequence shown in SEQ ID NO: 2, the polypeptide having thiosulphate sulphurtransferase activity.
 4. The process according to claim 1, wherein the expression of the gene coding for a polypeptide with the activity of a thiosulphate sulphurtransferase is increased by one or more measures selected from the group consisting of: a) the expression of the gene is under the control of a promoter which, in the microorganism used for the process, leads to an amplified expression compared with the starting strain; b) increasing the number of copies of the gene coding for a polypeptide with the activity of a thiosulphate sulphurtransferase compared with the starting strain; preferably by insertion of the gene into plasmids or by integration of the gene into the chromosome of the microorganism in several copies; c) the expression of the gene is effected using a ribosome binding site, which leads to an increased translation in the microorganism used for the process compared with the stalling strain; d) the expression of the gene is amplified by an optimization of the codon usage with respect to the microorganism used for the process; e) the expression of the gene is amplified by reduction of mRNA secondary structures in the mRNA transcribed by the gene; f) the expression of the gene is amplified by elimination of RNA polymerase terminators in the mRNA transcribed by the gene; and g) the expression of the gene is effected using mRNA-stabilizing sequences in the mRNA transcribed by the gene.
 5. The process according to claim 1, wherein the salt of dithiosulphuric acid is a salt selected from the group consisting of alkali metal salt, alkaline earth metal salt, ammonium salt and mixtures thereof, preferably ammonium salt.
 6. The process according to claim 1, wherein one the salt of sulphuric acid is a salt selected from the group consisting of alkali metal salt, alkaline earth metal salt, ammonium salt and mixtures thereof, preferably ammonium salt.
 7. The process according to claim 1, wherein during the fermentation the content of the salt of dithiosulphuric acid, based on the total content of inorganic sulphur in the medium and in the fermentation broth, is kept at at least 5 mol %.
 8. The process according to claim 1, wherein the sulphur-containing amino acid is L-methionine.
 9. The process according to claim 1, wherein the microorganism is of the genus Escherichia, preferably the species Escherichia coli.
 10. The process according to claim 1, wherein the microorganism is of the genus Corynebacterium, preferably the species Corynebacterium glutamicum.
 11. The process according to claim 1, wherein the microorganism is selected from the group consisting of Corynebacterium glutamicum with increased activity and/or expression of aspartate kinase and attenuation or deletion of the regulator protein McbR compared with the starting strain; and Escherichia coli with increased activity and/or expression of aspartate kinase and attenuation or deletion of the regulator protein MetJ compared with the starting strain. 12.-15. (canceled) 